Ramo Ana, Quílez Joaquín, Del Cacho Emilio, Sánchez-Acedo Caridad
Department of Animal Pathology, Faculty of Veterinary Sciences, University of Zaragoza, Miguel Servet 177, 50013 Zaragoza, Spain.
Department of Animal Pathology, Faculty of Veterinary Sciences, University of Zaragoza, Miguel Servet 177, 50013 Zaragoza, Spain.
Vet Parasitol. 2014 Oct 15;205(3-4):466-71. doi: 10.1016/j.vetpar.2014.08.025. Epub 2014 Sep 4.
A capillary electrophoresis (CE)-based DNA fragment analysis tool was optimized to identify in a single capillary the most common Cryptosporidium species and Cryptosporidium parvum GP60 alleles infecting domestic ruminants. For this purpose, a panel of genomic DNA samples including six Cryptosporidium species (C. parvum, C. bovis, C. ryanae, C. andersoni, C. ubiquitum, and C. hominis) and 18 C. parvum GP60 subtypes belonging to the subtype families IIa and IId was used. All these samples had been characterized previously by sequencing of SSU rRNA and GP60 genes. Isolates were re-amplified by PCR at these loci using sets of newly designed primers and subjected to CE. Fragment sizes were adjusted after comparison with sizes obtained by sequence analysis. The optimized CE-based approach provided fragments of different size for most Cryptosporidium species, but did not differentiate C. bovis and C. ryanae. Many of the GP60 subtypes (11/18) were also readily differentiated by CE, although overlapping in fragment sizes between IIa and IId subtypes was noticed. The CE-based tool was subsequently used to analyze Cryptosporidium isolates from naturally infected calves (n: 123) and lambs (n: 113) from farms in northern Spain. All isolates provided fragments typical of C. parvum. Fragment analysis at the GP60 locus differentiated a total of 10 alleles within isolates from calves (6 alleles) and lambs (8 alleles), with all but three alleles being host-associated. These findings support the validity of the optimized CE approach as a discriminatory and time- and cost-saving alternative to sequencing for identification of Cryptosporidium species and GP60 alleles in domestic ruminants.
一种基于毛细管电泳（CE）的DNA片段分析工具得到了优化，以便在单个毛细管中鉴定感染家养反刍动物的最常见隐孢子虫种类和微小隐孢子虫GP60等位基因。为此，使用了一组基因组DNA样本，包括六种隐孢子虫（微小隐孢子虫、牛隐孢子虫、雷氏隐孢子虫、安氏隐孢子虫、泛在隐孢子虫和人隐孢子虫）以及属于IIa和IId亚型家族的18种微小隐孢子虫GP60亚型。所有这些样本此前已通过小亚基核糖体RNA（SSU rRNA）和GP60基因测序进行了表征。使用新设计的引物对在这些位点的分离株进行PCR重新扩增，并进行毛细管电泳。与通过序列分析获得的大小进行比较后，调整片段大小。优化后的基于毛细管电泳的方法为大多数隐孢子虫种类提供了不同大小的片段，但无法区分牛隐孢子虫和雷氏隐孢子虫。许多GP60亚型（11/18）也能通过毛细管电泳轻松区分，不过注意到IIa和IId亚型之间的片段大小存在重叠。随后，基于毛细管电泳的工具被用于分析来自西班牙北部农场自然感染的犊牛（n = 123）和羔羊（n = 113）的隐孢子虫分离株。所有分离株都提供了微小隐孢子虫特有的片段。在GP60位点的片段分析在犊牛（6个等位基因）和羔羊（8个等位基因）的分离株中总共区分出10个等位基因，除了三个等位基因外，所有等位基因都与宿主相关。这些发现支持了优化后的毛细管电泳方法作为一种用于鉴定家养反刍动物中隐孢子虫种类和GP60等位基因的具有鉴别力且节省时间和成本的测序替代方法的有效性。
